Il-8 receptor antagonists

ABSTRACT

This invention relates to novel compounds of Formula (I) to (VII), and compositions thereof, useful in the treatment of disease states mediated by the chemokine, Interleukin-8 (IL-8).

FIELD OF THE INVENTION

This invention relates to novel sulfonamide substituted diphenyl ureacompounds, pharmaceutical compositions, processes for their preparation,and use thereof in treating IL-8, GROα, GROβ, GROγ, NAP-2, and ENA-78mediated diseases.

BACKGROUND OF THE INVENTION

Many different names have been applied to Interleukin-8 (IL-8), such asneutrophil attractant/activation protein-1 (NAP-1), monocyte derivedneutrophil chemotactic factor (MDNCF), neutrophil activating factor(NAF), and T-cell lymphocyte chemotactic factor. Interleukin-8 is achemoattractant for neutrophils, basophils, and a subset of T-cells. Itis produced by a majority of nucleated cells including macrophages,fibroblasts, endothelial and epithelial cells exposed to TNF, IL-1α,IL-1β or LPS, and by neutrophils themselves when exposed to LPS orchemotactic factors such as FMLP. M. Baggiolini et al., J. Clin. Invest.84, 1045 (1989); J. Schroder et al, J. Immunol. 139, 3474 (1987) and J.Immunol. 144, 2223 (1990); Strieter, et al., Science 243, 1467 (1989)and J. Biol. Chem. 264, 10621 (1989); Cassatella et al., J. Immunol.148, 3216 (1992).

GROα, GROβ, GROγ and NAP-2 also belong to the chemokine family. LikeIL-8 these chemokines have also been referred to by different names. Forinstance GROα, β, γ have been referred to as MGSAα, β and γ respectively(Melanoma Growth Stimulating Activity), see Richmond et al., J. CellPhysiology 129, 375 (1986) and Chang et al., J. Immunol 148, 451 (1992).All of the chemokines of the α-family which possess the ELR motifdirectly preceding the CXC motif bind to the IL-8 B receptor (CXCR2).

IL-8, GROα, GROβ, GROγ, NAP-2, and ENA-78 stimulate a number offunctions in vitro. They have all been shown to have chemoattractantproperties for neutrophils, while IL-8 and GROα have demonstratedT-lymphocytes, and basophilic chemotactic activity. In addition IL-8 caninduce histamine release from basophils from both normal and atopicindividuals. GRO-α and IL-8 can in addition, induce lysozomal enzymerelease and respiratory burst from neutrophils. IL-8 has also been shownto increase the surface expression of Mac-1 (CD11b/CD18) on neutrophilswithout de novo protein synthesis. This may contribute to increasedadhesion of the neutrophils to vascular endothelial cells. Many knowndiseases are characterized by massive neutrophil infiltration. As IL-8,GROα, GROβ, GROγ and NAP-2 promote the accumulation and activation ofneutrophils, these chemokines have been implicated in a wide range ofacute and chronic inflammatory disorders including psoriasis andrheumatoid arthritis, Baggiolini et al., FEBS Lett. 307, 97 (1992);Miller et al., Crit. Rev. Immunol. 12, 17 (1992); Oppenheimet al., Annu.Rev. Immunol. 9, 617 (1991); Seitz et al., J. Clin. Invest. 87, 463(1991); Miller et al., Am. Rev. Respir. Dis. 146, 427 (1992); Donnely etal., Lancet 341, 643 (1993). In addition the ELR chemokines (thosecontaining the amino acids ELR motif just prior to the CXC motif) havealso been implicated in angiostasis, Strieter et al., Science 258, 1798(1992).

In vitro, IL-8, GROα, GROβ, GROγ and NAP-2 induce neutrophil shapechange, chemotaxis, granule release, and respiratory burst, by bindingto and activating receptors of the seven-transmembrane, G-protein-linkedfamily, in particular by binding to IL-8 receptors, most notably theIL-8b receptor (CXCR2). Thomas et al., J. Biol. Chem. 266, 14839 (1991);and Holmes et al., Science 253, 1278 (1991). The development ofnon-peptide small molecule antagonists for members of this receptorfamily has precedent. For a review see R. Freidinger in: Progress inDrug Research, Vol. 40, pp. 33-98, Birkhauser Verlag, Basel 1993. Hence,the IL-8 receptor represents a promising target for the development ofnovel anti-inflammatory agents.

Two high affinity human IL-8 receptors (77% homology) have beencharacterized: IL-8Ra, which binds only IL-8 with high affinity, andIL-8Rb, which has high affinity for IL-8 as well as for GROα, GROβ, GROγand NAP-2. See Holmes et al., supra; Murphy et al., Science 253, 1280(1991); Lee et al., J. Biol. Chem. 267, 16283 (1992); LaRosa et al., J.Biol. Chem. 267, 25402 (1992); and Gayle et al., J. Biol. Chem. 268,7283 (1993).

There remains a need for treatment, in this field, for compounds, whichare capable of binding to the IL-8 a or b receptor. Therefore,conditions associated with an increase in IL-8 production (which isresponsible for chemotaxis of neutrophil and T-cells subsets into theinflammatory site) would benefit by compounds, which are inhibitors ofIL-8 receptor binding.

SUMMARY OF THE INVENTION

This invention provides for a method of treating a chemokine mediateddisease, wherein the chemokine is one which binds to an IL-8 a or breceptor and which method comprises administering an effective amount ofa compound selected from the following group of substituted3-phenylamino4H-1,2,4-benzothiadiazin-5-ol 1,1-dioxides:

-   7-chloro-3-[(2-chlorophenyl)amino]-4H-1,2,4-benzothiadiazin-5-ol    1,1-dioxide-   7-chloro-3-(cyclopentylamino)-4H-1,2,4-benzothiadiazin-5-ol    1,1-dioxide-   7-chloro-3-[(2,3-dichlorophenyl)amino]-4H-1,2,4-benzothiadiazin-5-ol    1,1-dioxide-   3-[(2-chlorophenyl)amino]-7-nitro-4H-1,2,4-benzothiadiazin-5-ol    1,1-dioxide-   3-[(2-bromophenyl)amino]-7-nitro-4H-1,2,4-benzothiadiazin-5-ol    1,1-dioxide-   7-nitro-3-{[2-(phenyloxy)phenyl]amino}-4H-1,2,4-benzothiadiazin-5-ol    1,1-dioxide-   3-[(2-chloro-3-fluorophenyl)amino]-7-nitro-4H-1,2,4-benzothiadiazin-5-ol    1,1-dioxide-   N-(2-chlorophenyl)-5-(methyloxy)-4H-1,2,4-benzothiadiazin-3-amine    1,1-dioxide and pharmaceutically acceptable salts thereof

SYNTHETIC EXAMPLES Example 1

7-chloro-3-[(2-chlorophenyl)amino]-4H-1,2,4-benzothiadiazin-5-ol1,1-dioxide

1) 4-Chloro-2-(methyloxy)aniline

A suspension of 2-nitro-5-chloroanisole (7.00 g), zinc dust (12.5 g) andacetic acid (7.8 ml) in ethanol (100 ml) was refluxed for 30-60 mins.The suspension was then filtered through celite and washed with ethylacetate. The filtrate was concentrated to give a gray solid, which wasredissolved in ethyl acetate (250 ml) and washed with water, saturatedaqueous sodium bicarbonate solution. The organic phase was dried overNa₂SO₄. The solvent was evaporated to give the title compound as a brownoil (4.65 g, 80%). LC/MS: m/z 158 (M+H).

1b) 7-chloro-5-(methyloxy)-2H-1,2,4-benzothiadiazin-3(4H)-one1,1-dioxide

A solution of 4-Chloro-2-(methyloxy)aniline (4.65 g) in nitropropane (5mL) was added to a solution of chlorosulfonyl isocyanate (3.16 mL) innitropropane (45 mL) at −40° C. for 5 mins. The reaction mixture wasallowed to warm up to 0° C. Aluminum chloride (4.94 g) was added atonce, resulting in a clear solution. The reaction mixture was heated at110° C. for 20 mins. After cooling down to room temperature, thereaction mixture was poured on ice-water mixture. The precipitate wasfiltered off and washed with water, dried in vacuo to give brown solid(4.0 g, 52%). LC/MS: m/z 263 (M+H).

1c)7-chloro-N-(2-chlorophenyl)-5-(methyloxy)-4H-1,2,4-benzothiadiazin-3-amine1,1-dioxide

Triflic anhydride (1.19 ml) was added to a solution of7-chloro-5-(methyloxy)-2H-1,2,4-benzothiadiazin-3(4H)-one 1,1-dioxide(740 mg) and pyridine (0.82 ml) in dichloromethane (15 mil) at −78° C.The reaction mixture was stirred for 1.5 hrs at this temperature beforequenching with aqueous amrnmonium chloride (5 ml). After warming up toroom temperature, the aqueous phase was extracted with methylenechloride (3×15 ml). The combined organic phase was dried over MgSO₄ andconcentrated to give the amidoyl triflate (0.86 g, 56%).

The resulting amidoyl triflate (250 mg) was dissolved in methylenechloride (15 ml). 2-Chloroaniline (0.20 ml) was added at −78° C. Thesolution was allowed to warm up to room temperature overnight. Thereaction mixture was diluted with dichloromethane (40 ml) and washedwith saturated aqueous sodium bicarbonate (50 ml). The aqueous phase wasextracted with dichloromethane (2×40 ml). The combined organic extractswere dried over MgSO₄ and evaporated in vacuo. The residue was dissolvedin DMSO (1.5 ml) and purified by Gilson preparatory HPLC to give thetitle compound (156 mg, 63%). LC/MS: m/z 372 (M+H).

1d) 7-chloro-3-[(2-chlorophenyl)amino]-4H-1,2,4-benzothiadiazin-5-ol1,1-doxide

7-chloro-N-(2-chlorophenyl)-5-(methyloxy)-4H-1,2,4-benzothiadiazin-3-amine1,1-dioxide (130 mg) was dissolved in methylenechloride (10 ml). BBr₃ (1ml) was added. The reaction mixture was refluxed for 6 hr. LCMS showedthat there was no starting material left. The reaction mixture waspurified by Gilson preparatory HPLC to give the title compound (91 mg,72%). LC/MS: m/z 358 (M+H).

Example 2

7-chloro-3-(cyclopentylamino)-4H-1,2,4-benzothiadiazin-5-ol 1,1-dioxide

Followed the general procedure outlined in Example 1c) and 1d),7-chloro-5-(methyloxy)-2H-1,2,4-benzothiadiazin-3(4H)-one 1,1-dioxide(250 mg) was treated with cyclopentylamine (163 mg, 1.91 mmol) followedby removal of protecting group with tribromoborane to give the titleproduct 122 mg (60%). ¹H NMR (CD₃OD) δ: 7.15 (1H, s), 6.93 (1H, s), 4.21(1H, m), 2.08 (2H, m), 1.72 (4H, m), 1.53 (2H,m).

Example 3

7-chloro-3-[(2,3-dichlorophenyl)amino]-4H-1,2,4-benzothiadiazin-5-ol1,1-dioxide

Followed the general procedure outlined in Example 1c) and 1d),7-chloro-5-(methyloxy)-4H-1,2,4-benzothiadiazin-3(4H)-one 1,1-dioxide(740 mg, 2.82 mmol) was treated with triflic anhydride (1.991 g, 7.06mmol) to give the crude amidoyl triflate 0.86 g. One portion of thiscrude material (316 mg, 0.81 mmol) was then reacted with2,3-dichloroanaline (260 mg, 1.62 mmol) followed by removal ofprotecting group with tribromoboron to yield the title prouduct 130 mg(41%). LC/MS: m/z 393 (M+H).

Example 4

3-[(2-chlorophenyl)amino]-7-nitro-4H-1,2,4-benzothiadiazin-5-ol1,1-dioxide

4a) 5-(methyloxy)-7-nitro-2H-1,2,4-benzothiadiazin-3(4H)-one 1,1-dioxide

Followed the general procedure outlined in Example 1b),2-methyloxy4-nitroaniline (4.97 g) was treated with chlorosulfonylisocyanate (3.17 ml) in nitroethane (45 ml) at −10° C. over 10 mins. Thein situ cyclization with aluminum chloride (5 g) yielded the desiredproduct (4.9 g, 60%). LC/MS: m/z 274 (M+H).

4b)N-(2-chlorophenyl)-5-(methyloxy)-7-nitro-4H-1,2,4-benzothiadiazin-3-amine1,1-dioxide

Followed the general procedure outlined in Example 1c),5-(methyloxy)-7-nitro-4H-1,2,4-benzothiadiazin-3(4H)-one 1,1-dioxide (70mg) was treated with triflic anhydride (0.13 mL) followed by reactingwith 2-Chloroaniline (0.034 ml) to give the title compound (33 mg, 33%).¹H NMR (CD₃OD) δ: 8.27 (1H, s), 8.16 (1H, d), 7.99 (1H, s), 7.49 (1H,d), 7.36 (1H, dd), 7.18 (1H, dd), 4.17 (3H, s).

4c) 3-[(2-chlorophenyl)amino]-7-nitro-4H-1,2,4-benzothiadiazin-5-ol1,1-dioxide

N-(2-chlorophenyl)-5-(methyloxy)-7-nitro-4H-1,2,4-benzothiadiazin-3-amine1,1-dioxide (120 mg) was dissolved in DMSO (3 ml). LiCl (60 mg) wasadded. The reaction mixture was heated at 150° C. for 8 hr. LCMC showedthat there was no starting meterial left. After cooling down to roomtemperature, LiCl was filtered off. The filtrate was purified by Gilsonpreparatory HPLC to give the title compound (92 mg, 80%). LC/MS: m/z 383(M+H).

Example 5

3-[(2-bromophenyl)amino]-7-nitro-4H-1,2,4-benzothiadiazin-5-ol1,1-dioxide

Followed the general procedure outlined in Example 1c) and 4c),5-(methyloxy)-7-nitro-2H-1,2,4-benzothiadiazin-3(4H)-one 1,1-dioxide(100 mg, 0.36 mmol) was treated with 2-bromoaniline (188 mg, 1.09 mmol)followed by removal of protecting group with LiCl to give the titleprouduct 22 mg (15%). ¹H NMR (DMSO) δ: 9.31 (1H, s), 7.93 (1H, s), 7.77(2H, m), 7.71 (1H, d), 7.44 (1H, t), 7.21 (1H, t).

Example 6

7-nitro-3-{[2-(phenyloxy)phenyl]amino}-4H-1,2,4-benzothiadiazin-5-ol1,1-dioxide

Followed the general procedure outlined in Example 1c) and 4c),5-(methyloxy)-7-nitro-2H-1,2,4-benzothiadiazin-3(4H)-one 1,1-dioxide(623 mg, 2.27 mmol) was treated with 2-phenoxyaniline (842 mg, 4.54mmol) followed by removal of protecting group with LiCl to give thetitle prouduct 122 mg (13%). ¹H NMR (DMSO) δ: 9.68 (1H, s), 8.16 (1H,d), 7.93 (1H, s), 7.81 (1H, br), 7.41 (1H, t), 7.26-7.09 (3H, m), 7.08(2H, d), 6.86 (1H, d).

Example 7

3-[(2-chloro-3-fluorophenyl)amino]-7-nitro-4H-1,2,4-benzothiadiazin-5-ol1,1-dioxide

Followed the general procedure outlined in Example 1c) and 4c),5-(methyloxy)-7-nitro-2H-1,2,4-benzothiadiazin-3(4H)-one 1,1-dioxide(500 mg, 1.82 mmol) was treated with 2-chloro-3-fluoro-aniline (532 mg,3.64 mmol) followed by removal of protecting group with LiCl to give thetitle prouduct 250 mg (35%). ¹H NMR (CD₃OD) δ: 8.04 (1H, s), 7.87 (1H,d), 7.71 (1H, s), 7.24 (1H, m), 7.03 (1H, m), 2.86 (1H, s).

Example 8

3-[(2-chlorophenyl)amino]-4H-1,24-benzothiadiazin-5-ol 1,1-dioxide

8a) N-(2-chlorophenyl)-5-(methyloxy)-4H-1,2,4-benzothiadiazin-3-amine1,1-dioxide

Followed the general procedure outlined in Example 1b) and 1c),2-methyloxyaniline (36.4 g, 29.55 mmol) was treated with chlorosulfonylisocyanate (5.16 g, 36.45 mmol) and trichloroaluminum (5 g, 37.44 mmol)to give the crude 5-(methyloxy)-2H-1,2,4-benzothiadiazin-3(4H)-one1,1-dioxide, which then reacted with 2-chloroaniline to yield the titleproduct 3.76 g (38%). ¹H NMR (CD₃OD) δ: 8.13 (1H, d), 7.43 (1H, d), 7.32(4H, m), 7.11 (1H, t), 4.08 (3H, s).

8b) 3-[(2-chlorophenyl)amino]-4H-1,2,4-benzothiadiazin-5-ol 1,1-dioxide

Followed the general procedure outlined in Example 4c),N-(2-chlorophenyl)-5-(methyloxy)-2H-1,2,4-benzothiadiazin-3-amine1,1-dioxide (0.5 g, 1.48 mmol) was treated with lithium chloride (0.5 g,11.8 mmol) to give the title compound 247 mg (52%). ¹H NMR (CD₃OD) δ:8.13 (1H, d), 7.43 (1H, d), 7.32 (1H, t), 7.21 (1H, d), 7.17 (2H, t),7.06 (1H, d).

METHOD OF TREATMENT

The compounds of Formula (I), or a pharmaceutically acceptable saltthereof can be used in the manufacture of a medicine for theprophylactic or therapeutic treatment of any disease state in a human,or other mammal, which is exacerbated or caused by excessive orunregulated IL-8 cytokine production by such mammal's cell, such as butnot limited to monocytes and/or macrophages, or other chemokines whichbind to the IL-8 a or b receptor, also referred to as the type I or typeII receptor.

Accordingly, the present invention provides a method of treating achemokine mediated disease, wherein the chemokine is one which binds toan IL-8 α or β receptor and which method comprises administering aneffective amount of a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof. In particular, the chemokines are IL-8, GROα,GROβ, GROγ, NAP-2 or ENA-78.

The compounds of Formula (I) are administered in an amount sufficient toinhibit cytokine function, in particular IL-8, GROα, GROβ, GROγ, NAP-2or ENA-78, such that they are biologically regulated down to normallevels of physiological function, or in some case to subnormal levels,so as to ameliorate the disease state. Abnormal levels of IL-8, GROα,GROβ, GROγ, NAP-2 or ENA-78 for instance in the context of the presentinvention, constitute: (i) levels of free IL-8 greater than or equal to1 picogram per mL; (ii) any cell associated IL-8, GROα, GROβ, GROγ,NAP-2 or ENA-78 above normal physiological levels; or (iii) the presenceof IL-8, GROα, GROβ, GROγ, NAP-2 or ENA-78 above basal levels in cellsor tissues in which IL-8, GRα, GROβ, GROγ, NAP-2 or ENA-78 respectively,is produced.

The compounds of Formula (I), in generally have been shown to have alonger t_(1/2) and improved oral bioavailabilty over the compoundsdisclosed in WO 96/25157 and WO 97/29743 whose disclosures areincorporated herein by reference.

There are many disease states in which excessive or unregulated IL-8production is implicated in exacerbating and/or causing the disease.Chemokine mediated diseases include psoriasis, atopic dermatitis,arthritis (either osteo- or rheumatoid), asthma, chronic obstructivepulmonary disease, adult respiratory distress syndrome, inflammatorybowel disease, Crohn's disease, ulcerative colitis, stroke, septicshock, endotoxic shock, gram negative sepsis, toxic shock syndrome,cardiac and renal reperfusion injury, glomerulonephritis, thrombosis,graft vs. host reaction, alzheimers disease, allograft rejections,malaria, restinosis, angiogenesis, atherosclerosis, osteoporosis,gingivitis, viral diseases such as rhinovirus or undesired hematopoieticstem cell release.

These diseases are primarily characterized by massive neutrophilinfiltration, T-cell infiltration, or neovascular growth, and areassociated with increased IL-8, GROα, GROβ, GROγ, NAP-2 or ENA-78production which is responsible for the chemotaxis of neutrophils intothe inflammatory site or the directional growth of endothelial cells. Incontrast to other inflammatory cytokines (IL-1, TNF, and IL-6), IL-8,GROα, GROβ, GROγ, NAP-2 or ENA-78 have the unique property of promotingneutrophil chemotaxis, enzyme release including but not limited toelastase release as well as superoxide production and activation. Theα-chemokines but particularly, GROα, GROβ, GROγ, NAP-2 or ENA-78,working through the IL-8 type I or II receptor can promote theneovascularization of tumors by promoting the directional growth ofendothelial cells. Therefore, the inhibition of IL-8 induced chemotaxisor activation would lead to a direct reduction in the neutrophilinfiltration.

Recent evidence also implicates the role of chemokines in the treatmentof HIV infections, Littleman et al., Nature 381, pp. 661 (1996) and Koupet al., Nature 381, pp. 667 (1996).

Present evidence also indicates the use of IL-8 inhibitors in thetreatment of atherosclerosis. The first reference, Boisvert et al., J.Clin. Invest, 1998, 101:353-363 shows, through bone marrowtransplantation, that the absence of IL-8 receptors on stem cells (and,therefore, on monocytes/macrophages) leads to a reduction in thedevelopment of atherosclerotic plaques in LDL receptor deficient mice.Additional supporting references are: Apostolopoulos, et al.,Arterioscler. Thromb. Vasc. Biol. 1996, 16:1007-1012; Liu, et al.,Arterioscler. Thromb. Vasc. Biol, 1997, 17:317-323; Rus, et al.,Atherosclerosis. 1996, 127:263-271.; Wang et al., J. Biol. Chem. 1996,271:8837-8842; Yue, et al., Eur. J. Pharmacol. 1993, 240:81-84; Koch, etal., Am. J. Pathol., 1993, 142:1423-1431.; Lee, et al., Immunol. Lett.,1996, 53, 109-113.; and Terkeltaub et al., Arterioscler. Thromb., 1994,14:47-53.

The present invention also provides for a means of treating, in an acutesetting, as well as preventing, in those individuals deemed susceptibleto, CNS injuries by the chemokine receptor antagonist compounds ofFormula (I).

CNS injuries as defined herein include both open or penetrating headtrauma, such as by surgery, or a closed head trauma injury, such as byan injury to the head region. Also included within this definition isischemic stroke, particularly to the brain area.

Ischemic stroke may be defined as a focal neurologic disorder thatresults from insufficient blood supply to a particular brain area,usually as a consequence of an embolus, thrombi, or local atheromatousclosure of the blood vessel. The role of inflammatory cytokines in thisarea has been emerging and the present invention provides a mean for thepotential treatment of these injuries. Relatively little treatment, foran acute injury such as these has been available.

TNF-α is a cytokine with proinflammatory actions, including endothelialleukocyte adhesion molecule expression. Leukocytes infiltrate intoischemic brain lesions and hence compounds which inhibit or decreaselevels of TNF would be useful for treatment of ischemic brain injury.See Liu et al., Stroke, Vol. 25., No. 7, pp. 1481-88 (1994) whosedisclosure is incorporated herein by reference.

Models of closed head injuries and treatment with mixed 5-LO/CO agentsis discussed in Shohami et al., J. of Vaisc & Clinical Physiology andPharmacology, Vol. 3, No. 2, pp. 99-107 (1992) whose disclosure isincorporated herein by reference. Treatment which reduced edemaformation was found to improve functional outcome in those animalstreated.

The compounds of Formula (I) are administered in an amount sufficient toinhibit IL-8, binding to the IL-8 a or b receptors, from binding tothese receptors, such as evidenced by a reduction in neutrophilchemotaxis and activation. The discovery that the compounds of Formula(I) are inhibitors of IL-8 binding is based upon the effects of thecompounds of Formulas (I) in the in vitro receptor binding assays whichare described herein. The compounds of Formula (I) have been shown to beinhibitors of type II IL-8 receptors.

As used herein, the term “IL-8 mediated disease or disease state” refersto any and all disease states in which IL-8, GROα, GROβ, GROγ, NAP-2 orENA-78 plays a role, either by production of IL-8, GROα, GROβ, GROγ,NAP-2 or ENA-78 themselves, or by IL-8, GROα, GROβ, GROγ, NAP-2 orENA-78 causing another monokine to be released, such as but not limitedto IL-1, IL-6 or TNF. A disease state in which, for instance, IL-1 is amajor component, and whose production or action, is exacerbated orsecreted in response to IL-8, would therefore be considered a diseasestate mediated by IL-8.

As used herein, the term “chemokine mediated disease or disease state”refers to any and all disease states in which a chemokine which binds toan IL-8 a or □ receptor plays a role, such as but not limited to IL-8,GROα, GRO-β, GROγ, NAP-2or ENA-78. This would include a disease state inwhich, IL-8 plays a role, either by production of IL-8 itself, or byIL-8 causing another monokine to be released, such as but not limited toIL-1, IL-6 or TNF. A disease state in which, for instance, IL-1 is amajor component, and whose production or action, is exacerbated orsecreted in response to IL-8, would therefore be considered a diseasestated mediated by IL-8.

As used herein, the term “cytokine” refers to any secreted polypeptidethat affects the functions of cells and is a molecule which modulatesinteractions between cells in the immune, inflammatory or hematopoieticresponse. A cytokine includes, but is not limited to, monokiries andlymphokines, regardless of which cells produce them. For instance, amonokine is generally referred to as being produced and secreted by amononuclear cell, such as a macrophage and/or monocyte. Many other cellshowever also produce monokines, such as natural killer cells,fibroblasts, basophils, neutrophils, endothelial cells, brainastrocytes, bone marrow stromal cells, epideral keratinocytes andB-lymphocytes. Lymphokines are generally referred to as being producedby lymphocyte cells. Examples of cytokines include, but are not limitedto, Interleukin-1 (IL-1), Interleukin-6 (IL-6), Interleukin-8 (IL-8),Tumor Necrosis Factor-alpha (TNF-α) and Tumor Necrosis Factor beta(TNF-β).

As used herein, the term “chemokine” refers to any secreted polypeptidethat affects the functions of cells and is a molecule which modulatesinteractions between cells in the immune, inflammatory or hematopoieticresponse, similar to the term “cytokine” above. A chemokine is primarilysecreted through cell transmembranes and causes chemotaxis andactivation of specific white blood cells and leukocytes, neutrophils,monocytes, macrophages, T-cells, B-cells, endothelial cells and smoothmuscle cells. Examples of chemokines include, but are not limited toIL-8, GRO-α, GRO-β, GRO-γ, NAP-2, ENA-78, IP-10, MIP-1α, MIP-β, PF4, andMCP 1, 2, and 3.

In order to use a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof in therapy, it will normally be formulated intoa pharmaceutical composition in accordance with standard pharmaceuticalpractice. This invention, therefore, also relates to a pharmaceuticalcomposition comprising an effective, non-toxic amount of a compound ofFormula (I) and a pharmaceutically acceptable carrier or diluent.

Compounds of Formula (I), pharmaceutically acceptable salts thereof andpharmaceutical compositions incorporating such may conveniently beadministered by any of the routes conventionally used for drugadministration, for instance, orally, topically, parenterally or byinhalation. The compounds of Formula (I) may be administered inconventional dosage forms prepared by combining a compound of Formula(I) with standard pharmaceutical carriers according to conventionalprocedures. The compounds of Formula (I) may also be administered inconventional dosages in combination with a known, second therapeuticallyactive compound. These procedures may involve mixing, granulating andcompressing or dissolving the ingredients as appropriate to the desiredpreparation. It will be appreciated that the form and character of thepharmaceutically acceptable character or diluent is dictated by theamount of active ingredient with which it is to be combined, the routeof administration and other well-known variables. The carrier(s) must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof.

The pharmaceutical carrier employed may be, for example, either a solidor liquid. Exemplary of solid carriers are lactose, terra alba, sucrose,talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acidand the like. Exemplary of liquid carriers are syrup, peanut oil, oliveoil, water and the like. Similarly, the carrier or diluent may includetime delay material well known to the art, such as glycerylmono-stearate or glyceryl distearate alone or with a wax.

A wide variety of pharmaceutical forms can be employed. Thus, if a solidcarrier is used, the preparation can be tableted, placed in a hardgelatin capsule in powder or pellet form or in the form of a troche orlozenge. The amount of solid carrier will vary widely but preferablywill be from about 25 mg to about 1 g. When a liquid carrier is used,the preparation will be in the form of a syrup, emulsion, soft gelatincapsule, sterile injectable liquid such as an ampule or nonaqueousliquid suspension.

Compounds of Formula (I) may be administered topically, that is bynon-systemic administration. This includes the application of a compoundof Formula (I) externally to the epidermis or the buccal cavity and theinstillation of such a compound into the ear, eye and nose, such thatthe compound does not significantly enter the blood stream. In contrast,systemic administration refers to oral, intravenous, intraperitoneal andintramuscular administration.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of inflammation such as liniments, lotions, creams, ointmentsor pastes, and drops suitable for administration to the eye, ear ornose. The active ingredient may comprise, for topical administration,from 0.001% to 10% w/w, for instance from 1% to 2% by weight of theFormulation. It may however comprise as much as 10% w/w but preferablywill comprise less than 5% w/w, more preferably from 0.1% to 1% w/w ofthe Formulation.

Lotions according to the present invention include those suitable forapplication to the skin or eye. An eye lotion may comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those for the preparation of drops. Lotions orliniments for application to the skin may also include an agent tohasten drying and to cool the skin, such as an alcohol or acetone,and/or a moisturizer such as glycerol or an oil such as castor oil orarachis oil.

Creams, ointments or pastes according to the present invention aresemi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the active ingredient infinely-divided or powdered form, alone or in solution or suspension inan aqueous or non-aqueous fluid, with the aid of suitable machinery,with a greasy or non-greasy base. The base may comprise hydrocarbonssuch as hard, soft or liquid paraffin, glycerol, beeswax, a metallicsoap; a mucilage; an oil of natural origin such as almond, corn,arachis, castor or olive oil; wool fat or its derivatives or a fattyacid such as steric or oleic acid together with an alcohol such aspropylene glycol or a macrogel. The formulation may incorporate anysuitable surface active agent such as an anionic, cationic or non-ionicsurfactant such as a sorbitan ester or a polyoxyethylene derivativethereof. Suspending agents such as natural gums, cellulose derivativesor inorganic materials such as silicaceous silicas, and otheringredients such as lanolin, may also be included.

Drops according to the present invention may comprise sterile aqueous oroily solutions or suspensions and may be prepared by dissolving theactive ingredient in a suitable aqueous solution of a bactericidaland/or fungicidal agent and/or any other suitable preservative, andpreferably including a surface active agent. The resulting solution maythen be clarified by filtration, transferred to a suitable containerwhich is then sealed and sterilized by autoclaving or maintaining at98-100° C. for half an hour. Alternatively, the solution may besterilized by filtration and transferred to the container by an aseptictechnique. Examples of bactericidal and fungicidal agents suitable forinclusion in the drops are phenylmercuric nitrate or acetate (0.002%),benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%).Suitable solvents for the preparation of an oily solution includeglycerol, diluted alcohol and propylene glycol.

Compounds of formula (I) may be administered parenterally, that is byintravenous, intramuscular, subcutaneous intranasal, intrarectal,intravaginal or intraperitoneal administration. The subcutaneous andintramuscular forms of parenteral administration are generallypreferred. Appropriate dosage forms for such administration may beprepared by conventional techniques. Compounds of Formula (I) may alsobe administered by inhalation, that is by intranasal and oralinhalation. administration. Appropriate dosage forms for suchadministration, such as an aerosol formulation or a metered doseinhaler, may be prepared by conventional techniques.

For all methods of use disclosed herein for the compounds of Formula (I)the daily oral dosage regimen will preferably be from about 0.01 toabout 80 mg/kg of total body weight. The daily parenteral dosage regimenabout 0.001 to about 80 mg/kg of total body weight. The daily topicaldosage regimen will preferably be from 0.1 mg to 150 mg. administeredone to four, preferably two or three times daily. The daily inhalationdosage regimen will preferably be from about 0.01 mg/kg to about 1 mg/kgper day. It will also be recognized by one of skill in the art that theoptimal quantity and spacing of individual dosages of a compound ofFormula (I) or a pharmaceutically acceptable salt thereof will bedetermined by the nature and extent of the condition being treated, theform, route and site of administration, and the particular patient beingtreated, and that such optimums can be determined by conventionaltechniques. It will also be appreciated by one of skill in the art thatthe optimal course of treatment, i.e., the number of doses of a compoundof Formula (I) or a pharmaceutically acceptable salt thereof given perday for a defined number of days, can be ascertained by those skilled inthe art using conventional course of treatment determination tests.

The invention will now be described by reference to the followingbiological examples which are merely illustrative and are not to beconstrued as a limitation of the scope of the present invention.

BIOLOGICAL EXAMPLES

The IL-8, and GRO-α chemokine inhibitory effects of compounds of thepresent invention are determined by the following in vitro assay:

Receptor Binding Assays:

[¹²⁵¹I] IL-8 (human recombinant) is obtained from Amersham Corp.,Arlington Heights, Ill., with specific activity 2000 Ci/mmol. GRO-α isobtained from NEN—New England Nuclear. All other chemicals are ofanalytical grade. High levels of recombinant human IL-8 type a and breceptors were individually expressed in Chinese hamster ovary cells asdescribed previously (Holmes, et al., Science, 1991, 253, 1278). TheChinese hamster ovary membranes were homogenized according to apreviously described protocol (Haour, et al., J. Biol. Chem., 249 pp2195-2205 (1974)). Except that the homogenization buffer is changed to10 mM Tris-HCl, 1 mM MgSO₄, 0.5 mM EDTA (ethylene-diaminetetra-aceticacid), 1 mM PMSF (α-toluenesulphonyl fluoride), 0.5 mg/L Leupeptin, pH7.5. Membrane protein concentration is determined using Pierce Co.micro-assay kit using bovine serum albumin as a standard. All assays areperformed in a 96-well micro plate format. Each reaction mixturecontains ¹²⁵I IL-8 (0.25 nM) or ¹²⁵¹I GRO-α and 0.5 μg/mL of IL-8Ra or1.0 μg/mL of IL-8Rb membranes in 20 mM Bis-Trispropane and 0.4 mM TrisHCl buffers, pH 8.0, containing 1.2 mM MgSO₄, 0.1 mM EDTA, 25 mM Na and0.03% CHAPS. In addition, drug or compound of interest is added whichhas been pre-dissolved in DMSO so as to reach a final concentration ofbetween 0.01 nM and 100 uM. The assay is initiated by addition of¹²⁵I-IL-8. After 1 hour at room temperature the plate is harvested usinga Tomtec 96-well harvester onto a glass fiber filtermat blocked with 1%polyethylenimine/0.5% BSA and washed 3 times with 25 mM NaCl, 10 mMTrisHCl, 1 mM MgSO₄, 0.5 mM EDTA, 0.03% CHAPS, pH 7.4. The filter isthen dried and counted on the Betaplate liquid scintillation counter.The recombinant IL-8 Ra, or Type I, receptor is also referred to hereinas the non-permissive receptor and the recombinant IL-8 Rb, or Type II,receptor is referred to as the permissive receptor.

Representative compounds of Formula (I), Examples 1 to 106 haveexhibited positive inhibitory activity in this assay at IC₅₀ levels<30uM.

Chemotaxis Assay:

The in vitro inhibitory properties of these compounds are determined inthe neutrophil chemotaxis assay as described in Current Protocols inImmunology, vol. I, Suppl 1, Unit 6.12.3., whose disclosure isincorporated herein by reference in its entirety. Neutrophils whereisolated from human blood as described in Current Protocols inImmunology Vol. I, Suppl 1 Unit 7.23.1, whose disclosure is incorporatedherein by reference in its entirety. The chemoattractants IL-8, GRO-α,GRO-β, GRO-γ and NAP-2 are placed in the bottom chamber of a 48multiwell chamber (Neuro Probe, Cabin John, Md.) at a concentrationbetween 0.1 and 100 nM. The two chambers are separated by a 5 uMpolycarbonate filter. When compounds of this invention are tested, theyare mixed with the cells (0.001-1000 nM) just prior to the addition ofthe cells to the upper chamber. Incubation is allowed to proceed forbetween about 45 and 90 min at about 37° C. in a humidified incubatorwith 5% CO₂. At the end of the incubation period, the polycarbonatemembrane is removed and the top side washed, the membrane then stainedusing the Diff Quick staining protocol (Baxter Products, McGaw Park,Ill., USA). Cells which have chemotaxed to the chemokine are visuallycounted using a microscope. Generally, four fields are counted for eachsample, these numbers are averaged to give the average number of cellswhich had migrated. Each sample is tested in triplicate and eachcompound repeated at least four times. To certain cells (positivecontrol cells) no compound is added, these cells represent the maximumchemotactic response of the cells. In the case where a negative control(unstimulated) is desired, no chemokine is added to the bottom chamber.The difference between the positive control and the negative controlrepresents the chemotactic activity of the cells.

Elastase Release Assay:

The compounds of this invention are tested for their ability to preventElastase release from human neutrophils. Neutrophils are isolated fromhuman blood as described in Current Protocols in Immunology Vol. I,Suppl 1 Unit 7.23.1. PMNs 0.88×10⁶ cells suspended in Ringer's Solution(NaCl 118, KCl 4.56, NaHCO₃ 25, KH₂PO₄ 1.03, Glucose 11.1, HEPES 5 mM,pH 7.4) are placed in each well of a 96 well plate in a volume of 50 ul.To this plate is added the test compound (0.001-1000 nM) in a volume of50 ul, Cytochalasin B in a volume of 50 ul (20ug/ml) and Ringers bufferin a volume of 50 ul. These cells are allowed to warm (37° C., 5% CO2,95% RH) for 5 min before IL-8, GROα, GROβ, GROγ or NAP-2 at a finalconcentration of 0.01-1000 nM was added. The reaction is allowed toproceed for 45 min before the 96 well plate is centrifuged (800×g 5min.) and 100 ul of the supernatant removed. This supernatant is addedto a second 96 well plate followed by an artificial elastase substrate(MeOSuc-Ala-Ala-Pro-Val-AMC, Nova Biochem, La Jolla, Calif.) to a finalconcentration of 6 ug/ml dissolved in phosphate buffered saline.Immediately, the plate is placed in a fluorescent 96 well plate reader(Cytofluor 2350, Millipore, Bedford, Mass.) and data collected at 3 minintervals according to the method of Nakajima et al J. Biol. Chem. 2544027 (1979). The amount of Elastase released from the PMNs is calculatedby measuring the rate of MeOSuc-Ala-Ala-Pro-Val-AMC degradation.

TNF-α in Traumatic Brain Injury Assay

The present assay provides for examination of the expression of tumornecrosis factor MRNA in specific brain regions, which followexperimentally, induced lateral fluid-percussion traumatic brain injury(TBI) in rats. Adult Sprague-Dawley rats (n=42) were anesthetized withsodium pentobarbital (60 mg/kg, i.p.) and subjected to lateralfluid-percussion brain injury of moderate severity (2.4 atm.) centeredover the left temporaparietal cortex (n=18), or “sham” treatment(anesthesia and surgery without injury, n=18). Animals are sacrificed bydecapitation at 1, 6 and 24 hr. post injury, brains removed, and tissuesamples of left (injured) parietal cortex (LC), corresponding area inthe contralateral right cortex (RC), cortex adjacent to injured parietalcortex (LA), corresponding adjacent area in the right cortex (RA), lefthippocampus (LH) and right hippocampus (RH) are prepared. Total RNA areisolated and Northern blot hybridization is performed and quantitatedrelative to an TNF-α positive control RNA (macrophage=100%). A markedincrease of TNF-α mRNA expression is observed in LH (104±17% of positivecontrol, p<0.05 compared with sham), LC (105±21%, p<0.05) and LA (69±8%,p<0.01) in the traumatized hemisphere 1 hr. following injury. Anincreased TNF-α mRNA expression is also observed in LH (46±8%, p<0.05),LC (30±3%, p<0.01) and LA (32±3%, p<0.01) at 6 hr which resolves by 24hr following injury. In the contralateral hemisphere, expression ofTNF-α mRNA is increased in RH (46±2%, p<0.01), RC (4±3%) and RA (22±8%)at 1 hr and in RH (28±11%), RC (7±5%) and RA (26±6%, p<0.05) at 6 hr butnot at 24 hr following injury. In sham (surgery without injury) or naiveanimals, no consistent changes in expression of TNF-α mRNA are observedin any of the 6 brain areas in either hemisphere at any times. Theseresults indicate that following parasagittal fluid-percussion braininjury, the temporal expression of TNF-α mRNA is altered in specificbrain regions, including those of the non-traumatized hemisphere. SinceTNF-α is able to induce nerve growth factor (NGF) and stimulate therelease of other cytokines from activated astrocytes, thispost-traumatic alteration in gene expression of TNF-α plays an importantrole in both the acute and regenerative response to CNS trauma.

CNS Injury Model for IL-1β mRNA

This assay characterizes the regional expression of interleukin-1β(IL-1β) mRNA in specific brain regions following experimental lateralfluid-percussion traumatic brain injury (TBI) in rats. AdultSprague-Dawley rats (n=42) are anesthetized with sodium pentobarbital(60 mg/kg, i.p.) and subjected to lateral fluid-percussion brain injuryof moderate severity (2.4 atm.) centered over the left temporaparietalcortex (n=18), or “sham” treatment (anesthesia and surgery withoutinjury). Animals are sacrificed at 1, 6 and 24 hr. post injury, brainsremoved, and tissue samples of left (injured) parietal cortex (LC),corresponding area in the contralateral right cortex (RC), cortexadjacent to injured parietal cortex (LA), corresponding adjacent area inthe right cortex (RA), left hippocampus (LH) and right hippocampus (RH)are prepared. Total RNA is isolated and Northern blot hybridization wasperformed and the quantity of brain tissue IL-1β mRNA is presented aspercent relative radioactivity of IL-1β positive macrophage RNA whichwas loaded on the same gel. At 1 hr following brain injury, a marked andsignificant increase in expression of IL-1β mRNA is observed in LC(20.00.7% of positive control, n=6, p<0.05 compared with sham animal),LH (24.5±0.9%, p<0.05) and LA (21.5±3.1%, p<0.05) in the injuredhemisphere, which remained elevated up to 6 hr. post injury in the LC(4.0±0.4%, n=6, p<0.05) and LH (5.0±1.3%, p<0.05). In sham or naiveanimals, no expression of IL-1β mRNA is observed in any of therespective brain areas. These results indicate that following TBI, thetemporal expression of IL-1β mRNA is regionally stimulated in specificbrain regions. These regional changes in cytokines, such as IL-1β play arole in the post-traumatic.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

The above description fully discloses the invention including preferredembodiments thereof. Modifications and improvements of the embodimentsspecifically disclosed herein are within the scope of the followingclaims. Without further elaboration, it is believed that one skilled inthe art can, using the preceding description, utilize the presentinvention to its fullest extent. Therefore the Examples herein are to beconstrued as merely illustrative and not a limitation of the scope ofthe present invention in any way. The embodiments of the invention inwhich an exclusive property or privilege is claimed are defined asfollows.

1. A compound selected from the froup consisting of:7-chloro-3-[(2-chlorophenyl)amino]-4H-1,2,4-benzothiadiazin-5-ol1,1-dioxide 7-chloro-5-(methyloxy)-2H-1,2,4-benzothiadiazin-3(4H)-one1,1-dioxide7-chloro-N-(2-chlorophenyl)-5-(methyloxy)-4H-1,2,4-benzothiadiazin-3-amine1,1-dioxide7-chloro-3-[(2-chlorophenyl)amino]-4H-1,2,4-benzothiadiazin-5-ol1,1-dioxide 7-chloro-3-(cyclopentylamino)-4H-1,2,4-benzothiadiazin-5-ol1,1-dioxide7-chloro-3-[(2,3-dichlorophenyl)amino]-4H-1,2,4-benzothiadiazin-5-ol,1,1-dioxide3-[(2-chlorophenyl)amino]-7-nitro-4H-1,2,4-benzothiadiazin-5-ol1,1-dioxideN-(2-chlorophenyl)-5-(methyloxy)-7-nitro-4H-1,2,4-benzothiadiazin-3-amine1,1-dioxide3-[(2-chlorophenyl)amino]-7-nitro-4H-1,2,4-benzothiadiazin-5-ol1,1-dioxide3-[(2-bromophenyl)amino]-7-nitro-4H-1,2,4-benzothiadiazin-5-ol1,1-dioxide7-nitro-3-{[2-(phenyloxy)phenyl]amino}-4H-1,2,4-benzothiadiazin-5-ol1,1-dioxide3-[(2-chloro-3-fluorophenyl)amino]-7-nitro-4H-1,2,4-benzothiadiazin-5-ol1,1-dioxide 3-[(2-chlorophenyl)amino]-4H-1,2,4-benzothiadiazin-5-ol1,1-dioxideN-(2-chlorophenyl)-5-(methyloxy)-4H-1,2,4-benzothiadiazin-3-amine1,1-dioxide
 2. A pharmaceutical composition comprising a compoundaccording to claim 1 and a pharmaceutically acceptable carrier ordiluent.
 3. A method of treating a chemokine mediated disease, whereinthe chemokine binds to an IL-8 a or b receptor in a mammal, which methodcomprises administering to said mammal an effective amount of a compoundaccording to claim
 1. 4. The method according to claim 3 wherein themammal is afflicted with a chemokine mediated disease selected from thegroup consisting of psoriasis, atopic dermatitis, arthritis (eitheroseo- or rheumatoid), asthma, chronic obstructive pulmonary disease,adult respiratory distress syndrome, inflammatory bowel disease, Crohn'sdisease, ulcerative colitis, stroke, septic shock, endotoxic shock, gramnegative sepsis, toxic shock syndrome, cardiac and renal reperfusioninjury, glomerulonephritis, thrombosis, graft vs. host reaction,alzheimers disease, allograft rejections, malaria, restinosis,angiogenesis, atherosclerosis, osteoporosis, gingivitis, viral diseasessuch as rhinovirus and undesired hematopoietic stem cell release.